1. Field of Endeavor
The present invention relates generally to the fields of cell biology, medicine and pathology. More particularly, it concerns methods and compositions relating to PDK inhibitors and their use to treat diseases ranging from diabetes to cardiovascular disease and cancer.
2. Description of Related Art
The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to give rise to acetyl-CoA, and is the gate-keeping enzyme linking glycolysis and the Krebs cycle. The mammalian PDC is a 9.5 million-dalton protein machine organized about a 60-meric core consisting of dihydrolipoyl transacetylase (E2) and the E3-binding protein (E3BP), to which multiple copies of pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase (E2), dihydrolipoamide dehydrogenase (E3), as well as isoforms of pyruvate dehydrogenase kinase (PDKs 1-4) and pyruvate dehydrogenase phosphatase (PDPs 1-2) are attached through ionic interactions (Reed 2001). Due to its strategic location, the regulation of PDC activity is critical for glucose homeostasis and fuel selection in the glucose-fatty acid cycle (Randle 1995). The mammalian PDC is acutely regulated by reversible phosphorylation (Harris et al., 1997). The phosphorylation of PDC by PDK results in inactivation; and dephosphorylation by PDP restores PDC activity. When glucose levels are low during fasting, PDC is highly phosphorylated and inactive, so as to preserve the substrates (pyruvate, lactate and alanine) for gluconeogenesis (Randle 1995).
The PDKs are potential therapeutic targets because of increased PDK expression in disease states such as diabetes, cancer and heart failure. PDK4, but not PDK2, is drastically induced in muscle and heart in streptozotocin-induced diabetes (Wu et al., 1999), obesity (Rosa et al., 2003) and type 2 diabetes (Kuzuya et al., 2008), which attenuates PDC activity leading to reduced glucose oxidation. The accumulated evidence has established that the upregulation of PDK4 is mediated through the PPARα-FOXO3α-PGC-1α complex (Wu et al., 1999). The PDK2/PDK4 double knockout mice fed a high-fat diet show marked improvements in glucose tolerance and insulin sensitivity over wild-type mice on the same diet (Jeoung et al., 2012). The expression of PDK1 (Papandreou et al., 2006; Kim et al., 2006 and Kaplon et al., 2013), PDK2 (Michelakis et al., 2010), and PDK3 (Lu et al., 2008) is significantly elevated in certain cancers. Tyrosine phosphorylation of PDK1 with increased kinase activity is essential for tumor cell proliferation and hypoxia (Hitosugi et al., 2011). Inhibition of PDK activity with dichloroacetate (DCA) or siRNA promotes apoptosis in cancer cells and impedes tumor growth (Bonnet et al., 2007).
The classic PDK inhibitor DCA, an analogue of the PDC substrate pyruvate, has been used since early 1970 to inhibit PDK activity and increase the PDC flux, with concomitant reduction in glucose levels in animals (Whitehouse and Randle 1973). DCA exerts its inhibitory effects by binding to an allosteric site in the N-terminal domain of PDK isoforms (Kato et al., 2007 and Knoechel et al., 2006). However, DCA is a non-specific low-potency PDK inhibitor and requires high doses for its therapeutic effects (Jiang et al., 2013), which leads to peripheral neurological toxicity and tumor growth (Stacpoole et al., 1997). R-lipoic acid in mM concentrations abates PDK activity in vitro (Korotchkina et al., 2004), but its function as a PDK inhibitor in vivo is uncertain. Phenylbutyrate enhances PDC activity in vitro and in vivo (Ferriero et al., 2013); but the compound is a modest PDK inhibitor (Ki=0.3 mM) with multiple targets and diverse clinical applications (Iannitti et al., 2011). Dihydrolipoamide mimetics including AZD7545 (Mayers et al., 2003) and secondary amides of SDZ048-619 (Aicher et al., 2000) have also been developed. This family of compounds inhibits PDK2 activity by impeding PDK binding to the E2/E3BP core of PDC (Kato et al., 2008). Paradoxically, these dihydrolipoamide mimetics strongly stimulates PDC core-free PDK4 activity in vitro, which precludes these compounds as bona fide PDK inhibitors (Wynn et al., 2008). To date, there have been no effective PDK inhibitors for novel therapeutic approaches to cancer, obesity and type 2 diabetes as well as heart disease.
Mitochondrial PDK isoforms are members of the GHKL ATPase/kinase superfamily that includes DNA gyrase B, heat-shock protein 90 (Hsp90), histidine kinases CheA and EnvZ as well as the DNA-repair enzyme MutL (Dutta and Inouye 2000). Members of this superfamily share four conserved motifs (N-, G1-, G2- and G3-boxes) that build a unique Bergerat ATP-binding fold consisting of a four-stranded mixed β-sheet and three a helices, and is located in the C-terminal domains of PDK isoforms (Steussy et al., 2001 and Kato et al., 2005). This signature fold also contains a unique structural element known as the “ATP lid”, whose conformational change is coupled to ATP hydrolysis and protein-protein interactions (Kato et al., 2005).